The goal of this project since its inception has been to understand virus structures, as a foundation for describing molecular events in virus assembly and entry. This renewal concerns entry. Advances in electron microscopy, in live-cell imaging, and in single-molecule detection allow us now to add to our structural snapshots the dimension of time and the context of a cell. Some properties of double-strand RNA (dsRNA) viruses offer particular advantages for studying non-enveloped virus entry; the flaviviruses offer related advantages for analyzing viral membrane fusion. Structures determined during the last grant period give us the information with which to ask precise questions about molecular mechanism. (1) Non- enveloped viral entry. We can follow rotavirus entry by live-cell fluorescence microscopy with particles bearing spectrally distinct labels on VP4 (the membrane perforation protein) and VP7 (the Ca2+ sensor for outer-layer uncoating). We can also use electron cryotomography (cryoET) to obtain medium-resolution images of entering particles. We will combine these approaches with information from the structure of the virion to work out the mechanisms of vesicular uptake, membrane perforation, and inner-particle release into the cytosol -- the sequence of events shown by our studies so far. (2) Enveloped-virus membrane fusion. We have developed a single-particle assay, to follow the time course of individual hemifusion and fusion events. We have shown how we can interpret kinetics of fusion, studied at the single-particle level, to probe the mechanism by which fusion proteins catalyze merger of two membrane bilayers. We will use this approach to study fusion of the non-infectious, recombinant virus-like particles that can be prepared with dengue and West Nile virus envelope proteins. The properties of these particles, in particular the ease of generating particles with a mutated envelope protein, will allow us to analyze steps in fusion that have not previously been accessible to single-particle kinetic analysis.